A Preliminary Genetic Linkage Map of Louisiana Sugarcane Using AFLP, TRAP and SSR markers
Pan, Y.-B.1, A. Suman2, S. Thongthewee2, C. A. Kimbeng2, B. E. Scheffler3, M. P. Grisham1,
T. L. Tew1, W. H. White1, and Edward Richard, Jr.1
1USDA-ARS,
MSA, Sugarcane Research Laboratory, Houma, LA; 2Louisiana State University, Baton Rouge, LA; 3USDA-ARS, Mid South Area, Genomics and
Bioinformatics Research Unit, Stoneville, MS
SUMMARY
Preliminary linkage maps of a Louisiana sugarcane cultivar, LCP 85-384, and its female (CP 77-310) and male (CP 77-407)
parents were developed from a S1 or peudo-F2 population using AFLP, TRAP and SSR markers. The maps allowed the study on
segregation pattern in mapping population and chromosome pairing during meiosis. These preliminary S1 maps would provide an
important framework for mapping QTLs associated with sugar yield, fiber content, as well as pest response traits and searching
for DNA markers to be used in the breeding programs.
INTRODUCTION
Sugarcane is a tall perennial plant typically grown in tropical and sub-tropical climates for sugar production. Early in the 20th
century, hybridization between S. officinarum (2n=80) and its wild relative S. spontaneum (2n=40-128) in Java, and backcrosses
of the inter-specific hybrids with S. officinarum resulted in modern sugarcane cultivars that produced higher sugar yields with
disease resistance. However, despite its economic importance worldwide, the complexity of the sugarcane genome has limited
classical genetic studies due to coexistence of simplex and multiplex alleles, irregular chromosome numbers in various
homo(eo)logy groups, and the difficulty of controlled hybridization. The initial difficulty in mapping sugarcane polyploids using
molecular markers was also due to the inability to identify the genotypes of marker phenotypes where a large number of
genotypes for each marker phenotype existed in a segregating population. With the advent of several molecular marker systems in
recent times, the efficiency of developing sugarcane molecular linkage maps, QTL mapping, and map-based cloning has been
increased. Furthermore, efforts in unraveling the sugarcane genome remain promising with the development of theoretical aspects
of genetic mapping in polyploids by Wu et al. (1992) using single dose fragments (SDF).
LCP 85-384 is considered the most successful sugarcane cultivar in the Louisiana sugar industry. The sugar yields of LCP 85384 were superior over the sugar yields of previously grown varieties by about 25%. Since its release in 1993, LCP 85-384 was
grown on up to 91% of the Louisiana sugarcane acreage. The cultivar has also been used frequently as a parent in the Louisiana
sugarcane breeding programs. However, the cultivar recently became susceptible to sugarcane borer (Diatreae saccharelis), ratoon
stunting disease (Leifsonia xyli subsp. xyli), and common rust (Puccinia melanocephala). Therefore, a molecular linkage map of
LCP 85-384 would be useful to understand the coexistence of genomic components derived from its parents and to dissect the
genetic basis of the superior agronomic traits.
The objective of the study is to construct a preliminary linkage map of LCP85-384 using AFLP, SSR, and TRAP markers
based on the selfed progeny of LCP 85-384. The mapping population segregates in relation to various diseases and a number of
agronomic traits. It is the first step towards the subsequent identification of QTLs for important agronomic traits.
MATERIALS AND METHODS
Plant materials: S1 (or pseudo F2 progeny) progeny were used that were derived from a spatially controlled selffertilization of LCP 85-384, a BC4 progeny selected out of the cross (CP 77-310 x CP 77-407). More than 1000 true S1 seeds were
germinated and 700 seedlings were transplanted. 300 S1 progeny were randomly selected for this study. Through vegetative
propagation, the mapping population was replicated and randomly planted in two field plots for evaluations and agronomic data
collection in two consecutive years. An additional set has been maintained in form of single pot culture in the green house. CP 77310 and CP 77-407 were also included to develop the grandparent maps of the pseudo F2 population. Leave tissue were collected
from the plants in the green house, freeze-dried, and stored in a refrigerator until DNA extraction. Genomic DNA was extracted
using the Plant DNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. Concentrations of extracted DNA
were estimated by Nanodrop 1000 spectrophotometer (Nanodrop, Bethesda, MD) and the DNA samples were stored at minus
20oC.
DNA extraction & marker protocols: AFLP was performed with total of 64 primer pairs according to Vos et al. (1995)
with some modifications. Twelve TRAP primer pairs were used. Primer design and PCR protocol were previously described by
Alwala et al. (2006). The forward primers were based on the gene/EST sequences of sucrose synthase (SuSy), soluble acid
invertase (SAI), calcium dependent protein kinase (CDPK), sucrose phosphate synthase (SuPS), pyruvate orthophosphate dikinase
(PODK), and starch synthase (StSy). The genes SuSy, SAI, SuPS, PODK, and StSy are associated with sucrose metabolism
whereas CDPK is believed to be associated with cold tolerance. Nineteen SSR primer pairs were used following a high throughput
genotyping protocol described by Pan et al. (2007).
In addition, two preliminary linkage maps were also constructed for the grandparents of the S1 population. The markers used were
based on an ‘ao x oo’ (CP77-310) and ‘oo x ao’ (CP77-407) configuration (that is present in one grandparent and absent in the
other), which segregated in a 3:1 fashion in the S1 population. We also used markers which were present in both the parents, F1, and
segregating in a 3:1 ratio in the S1 population.
RESULTS
A total of 1,113 polymorphic markers were produced from genotyping 300 S1
Figure 1: Distribution of
progeny of the cultivar LCP85-384 with 64 AFLP, 12 TRAP, and 19 SSR primer
segregation ratios for 1,113
pairs. The number of polymorphic markers scored per primer pair ranged from 1
markers
to 32 with a mean of 12. The Mather’s criterion excluded 338 likely multiple
dose markers and retained a total of 773 (69.5%) provisional single dose
markers, including 650 AFLP (84%), 94 TRAP (12%), and 29 SSR markers
(4%). Of the 773markers, 224 (29%) markers did not fit the theoretical single
dose markers ratio (3:1) using the chi-square test at 5% level (Type I error).
However, only 32 of 224 markers remained departed from theoretical
expectations after the Bonferroni procedure (α=6.47 x10-5). The distribution of
simplex markers was skewed toward the lowest ratios (less than 6.7), which
implies that the majority (~70%) of the markers could be considered to be single dose markers (Fig 1). The 773 markers were
divided according to their grandparental origin. Markers that did not have information on grandparental origin were discarded from
the analysis. The female parent (CP 77-310) contributed 210 AFLP markers with a mean of 3.5, whereas the male parent (CP 77407) contributed 167 AFLP markers with a mean of 2.78. However, the AFLP markers found in both grandparents (‘ao x ao’) which
segregated in a 3:1 ratio in the S1 were 230 with a mean of 3.84. The 230 markers were appended to the grandparent-specific
markers, which yielded a total of 440 (210+230) for the female grandparent and 397 (167+230) markers for male grandparent. The
number of distorted markers recorded at 5% level of significance in CP 77-310 and CP 77-407 were 127 (29%) and 130 (32.7%),
respectively. After the Bonferroni procedure, the number of distorted markers in CP 77-310 was reduced from 127 to 19, whereas
the reduction in CP 77-407 was from 130 to 18.
Table 1: AFLP, TRAP, and SSR polymorphic markers used for constructing a genetic linkage map of LCP 85-384
Primer
pair
E32M47
E32M48
E32M49
E32M50
E32M59
E32M60
E32M61
E32M62
E33M47
E33M48
E33M49
E33M50
E33M59
E33M60
E33M61
E33M62
E36M47
E36M48
E36M49
E36M50
E36M59
E36M60
E36M61
E36M62
E37M47
E37M48
E37M49
E37M50
E37M59
E37M60
E37M61
E37M62
E38M47
#Markers
scored
21
16
28
20
15
21
27
10
9
8
12
15
20
21
18
21
26
14
31
9
32
26
28
17
15
14
15
12
14
11
15
8
7
Mather's Co-segregation groups covered
criterion
(6.7:1)
10
14
16
13
10
11
18
3
6
8
11
10
15
15
15
14
11
11
20
7
18
13
22
11
11
6
14
10
10
7
12
6
5
64 AFLP markers
8,19,37,43,44,48,49,61,75,88 (10)
2,3,15,20,34,36,37,46,54,55,63,83,93 (13)
5,16,18,27,28,34,42,48,49,53,77,80,82,84 (14)
2,17,18,51,67,72,73,75,78,90,96 (11)
6,13,27,31,36,43,44,92,100 (9)
1,6,10,17,22,49,62,65,96 (9)
4,5,12,13,23,27,33,36,45,48,54,62,70,71, 76, 88,91(17)
16,73,88 (3)
21,40,41,49,50 (5)
1,5,19,44,49,66,81,90 (8)
12,16,17,31,39,49,75,89,103 (9)
14,17,21,25,56,57,94,96,101,104 (10)
1,7,16,20,22,24,35,44,45,47,76,78,83,89 (14)
1,17,45,54,56,65,74,75,82,99,101 (11)
10,13,14,50,52,53,61,74,78,84,87,88,89 (13)
5,10,16,31,39,43,44,46,51,69,70,83,88 (13)
4,7,9,12,18,55,55,75,85,100,104 (10)
27,29,44,48,54,74,85 (7)
1,2,9,17,20,21,23,39,51,60,67,75,78,93,97,99,108 (17)
43,44,46,75,90,91,105 (7)
1,4,8,9,10,12,17,27,31,45,47,50,53,79,107 (15)
1,12,15,31,34,35,39,46,75,90,91 (11)
1,4,7,20,22,23,34,43,50,56,71,73,76,78,79,83,85,88,96 (19)
7,20,43,63,75,83,85,87,105 (9)
11,20,34,35,43,49,55,57 (8)
33,47,57,86,106,107 (6)
5,24,25,31,42,44,47,70,76,78 (10)
5,33,43,47,50,51,74,75,89,98,101 (11)
2,20,31,37,39,52,78,98 (8)
27,39,43,48,70,78 (6)
1,16,18,25,26,27,28,35,46,54,74,85,89 (13)
2,22,40,71,77,90 (6)
4,12,24,35,53 (5)
Primer
pair
E38M48
E38M49
E38M50
E38M59
E38M60
E38M61
E38M62
E39M47
E39M48
E39M49
E39M50
E39M59
E39M60
E39M61
E39M62
E40M47
E40M48
E40M49
E40M50
E40M59
E40M60
E40M61
E40M62
E41M47
E41M48
E41M49
E41M50
E41M59
E41M60
E41M61
E41M62
Sub-total
Mean
Range
#Markers
scored
Mather's
criterion
(6.7:1)
17
14
12
15
10
11
8
10
20
23
11
6
23
21
9
6
10
13
8
18
13
9
14
6
10
6
8
1
17
21
6
952
14.87
1- 32
14
10
10
14
10
11
8
6
14
13
8
6
13
13
4
5
7
10
5
11
8
7
9
3
8
4
6
1
9
16
4
650
10.15
1-22
Co-segregation groups covered
17,23,28,30,31,37,39,43,55,80,82,86 (12)
8,23,37,49,68,70,76,83 (8)
2,10,11,62,69,75,78,80,86, 97 (10)
3,4,12,20,36,46,54,56,83,84,97,102 (12)
20,21,25,39,52,78,83,94 (8)
1,24,34,36,40,44,55,73,89,93 (10)
11,17,28,47,73,75 (6)
1,39,61,81,88,102 (6)
10,11,20,21,25,26,30,39,49,58,80,81 (12)
7,16,18,23,34,50,51,52,53,80,82,85 (12)
4,35,37,70,92,94 (6)
12,30,45,59,91,93 (6)
1,4,6,15,22,23,32,47,70,76,84,98 (12)
4,10,20,21,46,57,58,63,81,83,83,95 (11)
15,37,52,77 (4)
20,24,51,79,80 (5)
2,43,68,77,92 (6)
2,4,6,16,27,31,44,60,91,108 (10)
2,40,81,85,87 (5)
1,3,39,60,64,77,84 (7)
9,18,34,39,41,65,80,90 (8)
44,45,58,81,91 (5)
1,12,16,17,18,23,50,86,103 (9)
22,78,80 (3)
1,20,37,51,57,74,84 (7)
6,52 (2)
42,43,59,76,85,106 (6)
14 (1)
4,7,14,31,32,42,64,78 (8)
2,11,12,15,16,17,23,42,47,80,81,83,84 (13)
1,8,15,38 (4)
Primer
pair
#Markers
scored
SuSy_R1
SuSy_R3
SuPS_R1
SuPS_R3
PODK_R1
PODK_R3
SAI_R1
SAI_R3
CDPK_R1
CDPK_R3
StSy_R1
StSy_R3
Sub-total
Mean
Range
9
6
15
6
8
8
7
4
10
12
5
10
100
8.34
4-15
119CG
1604SA
1751CL
18SA
278CS
31CUQ
334BS
336BS
36BUQ
486CG
569CS
597CS
7CUQ
CIR19
CIR29
CIR66
CIR74
703BS
CIR3
Sub-total
Mean
Range
2
4
4
4
3
3
2
3
2
3
3
4
3
4
5
3
3
5
1
61
3.2
1-5
Mather's Co-segregation groups covered
criterion
(6.7:1)
12 TRAP markers
9
2,42,43,45,58,71,72,85,98 (9)
5
21,24,53,71 (4)
15
4,37,38,45,48,81,92 (7)
6
2,27,31,44,90 (5)
7
4,10,15,37,42,45,81 (7)
7
16,20,45,52,82,87 (6)
6
31,45,59,75,86,100 (6)
4
6,8,47 (3)
8
1,16,20,46,56,66,84 (7)
12
1,8,21,24,37,39,48,81,84,99 (10)
5
12,20,33,73,83 (5)
10
24,37,39,55,74,75,83,85,95 (9)
94
7.84
4-15
19 SSR markers
1
70 (1)
2
4,5 (2)
2
44,49 (2)
3
12,23,40 (3)
2
10,11 (2)
2
10,11 (2)
1
13 (1)
2
10,11 (2)
1
20 (1)
1
0
3
49 (1)
1
64 (1)
1
73 (1)
2
70,97 (2)
3
27,29,75 (3)
1
27 (1)
1
93 (1)
0
0
0
0
29
1.52
0-3
Mapping of simplex markers onto co-segregation (CG) groups (Grivet et al. 1996) was implemented using JoinMap v3.0.
Linkages in coupling phase were detected using an upper recombination fraction threshold of 0.44 (maxr= {0.5 – z (α) √ 0.5 (10.5)/n}, where z (0.01) = 2.3264, and n=300) (Wu et al. 1992). Only coupling phase linkages were detected and included on the
linkage map. The non-simplex markers both in coupling and repulsion phases were ignored irrespective of their inheritance pattern
(Grivet et al., 1996). In order to avoid false linkages, multiple two-point linkage analyses were performed at LOD score ≥ 4.0.
Cosegregation groups (CGs), which correspond to a single chromosome among all the homo(eo)logous chromosomes, were
identified by grouping the linked markers. The genetic distances (in cM) between the markers on the map were computed from
recombination fractions using the Kosambi mapping function. The CGs were assembled into homologous groups based on the
alleles generated by the same SSR primer pair and information on the grandparental maps.
Figure 3: Female (CP 77-310) grandparent linkage map of the
sugarcane cultivar LCP 85-384 constructed from a self-progeny
population of 300 individuals. The map was constructed with a LOD
score > 4.0 and a recombination fraction of 0.44 using AFLP markers.
Only five CGs are shown here. The vertical bars indicate CGs with
markers in coupling phase linkages. The Kosambi map distances (cM)
marker names are indicated on the left and right sides, respectively, of
each CG. AFLP markers are denoted by ‘EM’. CGs were grouped into
HG based on S1map information. The CP 77-310-specific markers are
represented by two dots (··). The markers present in both parents and
segregated in S1 population are denoted by the † symbol. The marker
names with an asterisk (*) represent distorted markers.
Figure 4: Male (CP 77-407) grandparent linkage map of the sugarcane cultivar LCP
85-384 from a self-progeny population of 300 individuals. The map was constructed
with a LOD score > 4.0 and a recombination fraction of 0.44 using AFLP markers.
Only five CGs are shown here. The vertical bars indicate CGs with markers in
coupling phase linkages. The Kosambi map distances (cM) marker names are
indicated on the left and right sides, respectively, of each CG. AFLP markers are
denoted by ‘EM’. The CP 77-407-specific markers are represented by one dots (·).
The markers present in both parents and segregated in S1 population are denoted by
the † symbol. The marker names with an asterisk (*) represent distorted markers.
Homo(eo)logous groups (HGs): The co-dominance nature of SSR markers and the information on grandparental maps enabled the
CGs to be assembled into putative homo(eo)logous groups (HGs) (Fig. 2 for example). The AFLP markers belonging to a single
grandparental CG were distributed in more than one of the S1CGs. Indeed, they were either part of a large CG or belong to a HG.
From the final 108 CGs, a total of 31 CGs were assembled into 12 putative HGs using a total of 10 SSR loci (20 alleles), 5 CGs of
‘CP 77-310’ and 6 CGs of ‘CP 77-407’. HG-I was the biggest group with 6 CGs, where as the remaining HGs contained either 2 or 3
CGs. The CG-4, -5, 12, -23, and -40 in HG-I shared 3 SSR alleles from locus 18SA, and 2 SSR alleles from locus 1604SA. A
maximum of 3 loci (278CS, 336BS, and 31CUQ) which had consistent genomic position in CG-11 and CG-12 were grouped in HGII (not shown). CG-31, -32, and -33 were formed into HG-XII based on the CG310-39. The remaining CGs in the S1 map, either had
no SSR markers in common or the grandparental maps information, were considered as independent groups. On grand parental
linkage maps, CGs were also arranged into putative HGs using information from the S1 map. In ‘CP 77-407’ linkage map, a total of
seven HGs were found based on 7 CGs of the S1 linkage map. In contrast, four HGs were found in the ‘CP 77-310’ using the
information on three CGs of S1 linkage map. In both grandparental HGs, a maximum of two chromosomes were found in each HG.
The remaining CGs in both grandparental maps was considered as independent groups (Not shown).
Chromosomal paring, genome size, and genome coverage: Repulsion phase linkages were found in CGs of LCP 85-384 and its
parents. All together, recombination frequency estimates were computed for 597,529 (773*773), 193,600 (440*440), and 15,609
(397*397) pair-wise combinations for LCP 85-834, CP 77-310, and CP 77-407, respectively. A total of 4265:4306, 1639:1539, and
1362:1302 coupling to repulsion linkages were detected and confirmed the 1:1 ratio in LCP 85-384, CP 77-310 and CP 77-407 linkage
maps, respectively. Moreover, for each CG on the S1 and grandparental maps, the markers in C-C and R-R phase linkages confirmed the
ratio 1:1(χ2 at 0.05), which supports prevalence of disomic inheritance (preferential pairing). CGs on S1 map were not identified based on
chromosomal origin of ancestral species (S. officinarum and S. spontaneum), but by chromosomal origins of the grandparents. Out of 108
CGs in the S1 map, a total of 42 of CP 77-310 chromosomal origin, 44 of CP 77-310 chromosomal origin, and 9 were recombinant.
Thirteen CGs could not be marked with of grandparental specific chromosomal origins. Approximate genome sizes of LCP 85-384 and
its parents (CP 77-310 x CP 77-407) were estimated using the method followed by Aitken et al. (2005) and Hoarau et al. (2001). The
estimated genome size of LCP 85-384, 106 x 120 =12,720 cM, was obtained by multiplying the chromosome number (106) with the
average size of the longest CGs (120 cM). Likewise, the estimated genome sizes of CP 77-310 and CP 77-407 were 14,950 cM (115 x
130 cM), and 11,500 cM (115 x 100 cM), respectively. The ratio between the cumulative genome length and estimated genome length
indicated that approximately 42% (5384/12720), 23.2 % (3476/14950), and 24.1 % (2777/11500) of the LCP 85-384, CP 77-310, and CP
77-410 genomes, respectively, have been covered in this study.
DISCUSSION
Marker scoring, segregation analyses & linkage map construction: The AFLP- and TRAP- DNA fragments were
run on a LiCor 4300 DNA analyzer, while the SSR-DNA fragments were run on an ABI3730XL Genetic Analyzer. Amplified
fragments were MANUALLY scored as ‘1’ for the presence or ‘0’ for the absence. AFLP markers were denoted by ‘EM’ for
ECoRI–MseI primer pairs with band size as suffix using universal nomenclature where the numbers followed by each letter are
codes for a primer pair. TRAP markers were denoted by the codes for forward and reverse primers along with marker size as
suffix. SSR markers were indicated with its name and identity number from the Sugarcane Microsatellite Consortium along with
the allele size as suffix. Polymorphic markers were divided into three classes based on possible segregation ratios upon χ2 tests. In
the absence of segregation distortion and in the presence of disomic inheritance, single dose (simplex), double dose (duplex), and
triple dose (triplex) markers would segregate in 3:1, 15:1, and 63:1 ratios, respectively. Except for single dose markers, these ratios
would become complicated in case of polysomic inheritance. Therefore, only single dose (simplex) markers were included in the
linkage mapping because they are the most informative type (Wu et al. 1992; Grivet et al. 1996). In this study, the size of the
mapping population was confirmed to be large enough to differentiate the 3:1 segregating markers from all other non-simplex
markers. However, we retained all the markers that have a +/- ratio of lower than 6.7:1 (√3 x 15:1) to ensure the selection of only
simplex markers. It was done by choosing the smallest non-simplex marker ratio (15:1) to cull out all non-simplex markers that
are segregating in higher ratios. On those markers selected, segregation analysis was conducted again against the expected 3:1
Mendilian ratio using χ2 test (1 df) at 5% error level (Type I). In addition, a Bonferroni correction was also applied to limit the
experiment-wide error rate associated with multiple testing (Sokal and Rohlf, 1995). Critical χ2 values were calculated by dividing
the alpha (0.05) by the number of markers. We considered the distorted markers (α=6.47 x10-5) for linkage mapping and those
markers deviated from the theoretical expected ratio (3:1) even after the Bonferroni corrections were marked with asterisk.
Disclaimer: Product names and trademarks are mentioned to report
factually on available data; however, the USDA neither guarantees nor
warrants the standard of the product, and the use of the name by USDA
does not imply the approval of the product to the exclusion of others that
may also be suitable. The experiments reported comply with the current
laws of U.S.A.
A maximum detectable recombination threshold of 0.44 and LOD score values of ≥ 4.0 (one allowed error in 10,000 linkages) were
used in this study to give high confidence in the map by avoiding spurious linkages. However, the maximum detectable recombination
generally depends on the size of mapping population. The S1 map of the commercial hybrid ‘R570’ reported by Grivet et al. (1996) and
Hoarau et al. (2001) contained 96 CGs spanning 2,008 cM and 120 CGs spanning 5,849 cM, respectively. In this study, we have 108
CGs covering cumulative map length of 5,384 cM. The number of CGs observed in S1 map was close to the expected number of
chromosome number in LCP 85-384 (2n=106). In contrast, for the grandparental maps, CP 77-310 had 81 CGs spanning 3,476 cM and
‘CP77-407’ had 80 CGs covering 2,777cM. Our preliminary linkage maps of S1, female and male grandparents were not saturated and
covered only 42%, 23%, and 24% of the genome, correspondingly.
Figure 2: HG-I linkage map of Louisiana sugarcane cultivar ‘LCP 85-384’ from a self-progeny population of 300 individuals. The vertical bars indicate
CGs with markers in coupling phase linkages. The Kosambi map distances (cM) marker names are indicated on the left and right sides, respectively, of each
CG. CGs were grouped into HG based on SSR loci and information on grandparental maps. The SSR alleles responsible for in each HG are represented
bold. The grandparental specific markers are represented by one dot (·) or two dots (··) for CP77-407 and CP77-310, respectively. The markers present in
both parents and segregated in S1 population are denoted by the † symbol. Marker names with an asterisk (*) represent distorted markers.
S1 map: Out of the 773 simplex markers, 717 markers were assigned to 108 CGs (6 CGs on HG-I is shown in Fig. 2) with a
cumulative genome length of 5384 cM. Fifty six markers remained unlinked. The length of the CGs varied from 4cM (CG-102,
and -68) to 147cM (CG-39) with an average of 7.5 cM between any two adjacent markers.
Grandparent Maps: Information from only 60 AFLP markers was available for this analysis. A total of 440 markers in the
female grandparent (CP77-310) and 397 markers in the male grandparent (CP77-407) were used for the grandparental maps.
The CP77-310 map comprised of 391 linked markers that spread over 81 CGs with a cumulative genomic length of 3476 cM,
where 49 markers remained unlinked (Fig. 3 for example). In contrast, the CP77-407 map comprised of 339 markers spanning
80 CGs with a cumulative genome length of 2777 CM, while 58 markers remained unlinked (Fig. 4 for example).
The preliminary linakge maps in the current study has uneven marker distribution along the CGs. Other studies have shown that the
S. spontaneum protion of the genome is better mapped comparing to the S. officinarum portion of the genome. For this reason, some of
the CGS were probably dense. The uneven marker distribution on the linkage maps could also be due to the use of only single dose
markers and coupling phase linkages. These unsaturated linkage maps were also evident by the high number of unlinked markers
coupled with short CGs (have less than 3 markers per CG). A comparable number of unlinked markers were also reported by Aitken et
al. (2005) and Hoarau et al. (2001) while significantly higher numbers of unlinked markers was reported by Garcia et al. (2006) and
Alwala et al. (2008). The difference could be due to the number of progeny and type of the population used in these studies. Several
short CGs, which might be part of the larger CGs, could be consequential of using the higher LOD values (≥ 4.0). More markers are
needed in order to saturate the preliminary maps and therefore to make them amenable to the QTL discovery.
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